7-beta-hydroxysteroid dehydrogenase mutants and process for the preparation of ursodeoxycholic acid

A nucleic acid encoding a 7β-hydroxysteroid dehydrogenase (7β-HSDH) that catalyzes at least the stereospecific enzymatic reduction of a 7-ketosteroid to the corresponding 7-hydroxysteroid, wherein the enzyme comprises a mutation at position 64 of SEQ ID NO:2 or in the corresponding sequence positions of an amino acid sequence derived therefrom with at least 80% sequence identity to SEQ ID NO:2, and additionally has at least one mutation in the sequence motif VMVGRRE as per position 36 to 42 of SEQ ID NO:2 or in the corresponding sequence motif of an amino acid sequence derived therefrom with at least 80% sequence identity to SEQ ID NO:2;

• wherein the mutation at position 64 is the mutation R64X1, wherein X1 represents E, D, T, L, S, P, V, K, C, A, G, Q, F, W, I or Y;
• and
• wherein the enzyme shows the following property profile in comparison with the 7β-HSDH with SEQ ID NO:2:
• a) an increased specific activity (Vmax [U/mg]) for NAD(P)H in the enzymatic reduction of dehydrocholic acid (DHCA) with NAD(P)H as cofactor;
• and additionally optionally:
• b) an increased specific activity (Vmax [U/mg]) for DHCA in the enzymatic reduction of DHCA with NADPH as cofactor;
• c) a reduced substrate inhibition by DHCA;
• d) a modified cofactor specificity with respect to NADH and NADPH;
• and
• e) it being possible for these properties b), c) and d) to be present individually or in any combination.

A process for the enzymatic or microbial synthesis of 7β-hydroxysteroids, wherein the corresponding 7-ketosteroid is detected in the presence of a 7β-hydroxysteroid dehydrogenase (7β-HSDH) that catalyzes at least the stereospecific enzymatic reduction of a 7-ketosteroid to the corresponding 7-hydroxysteroid, wherein the enzyme comprises a mutation at position 64 of SEQ ID NO:2 or in the corresponding sequence positions of an amino acid sequence derived therefrom with at least 90% sequence identity to SEQ ID NO:2;

• wherein the mutation at position 64 is the mutation R64X1, wherein X1 represents E, D, T, L, S, P, V, K, C, A, G, Q, F, W, I or Y;
• and
• wherein the enzyme shows the following property profile in comparison with the 7β-HSDH with SEQ ID NO:2:
• a) an increased specific activity (Vmax [U/mg]) for NADPH in the enzymatic reduction of dehydrocholic acid (DHCA) with NADPH as cofactor;
• and additionally optionally:
• b) an increased specific activity (Vmax [U/mg]) for DHCA in the enzymatic reduction of DHCA with NADPH as cofactor;
• c) a reduced substrate inhibition by DHCA;
• d) a modified cofactor specificity with respect to NADH and NADPH;
• and
• e) it being possible for these properties b), c) and d) to be present individually or in any combination.

A process for the enzymatic or microbial synthesis of 7β-hydroxysteroids, wherein the corresponding 7-ketosteroid is detected in the presence of a 7β-hydroxysteroid dehydrogenase (7β-HSDH) that catalyzes at least the stereospecific enzymatic reduction of a 7-ketosteroid to the corresponding 7-hydroxysteroid, wherein the enzyme comprises a mutation at position 64 of SEQ ID NO:2, and additionally has at least one mutation in the sequence motif VMVGRRE as per position 36 to 42 of SEQ ID NO:2 or in the corresponding sequence positions of an amino acid sequence derived therefrom with at least 80% sequence identity to SEQ ID NO:2;

• wherein the mutation at position 64 is the mutation R64X1, wherein X1 represents E, D, T, L, S, P, V, K, C, A, G, Q, F, W, I or Y;
• and
• wherein the enzyme shows the following property profile in comparison with the 7β-HSDH with SEQ ID NO:2:
• a) an increased specific activity (Vmax [U/mg]) for NADPH in the enzymatic reduction of dehydrocholic acid (DHCA) with NADPH as cofactor;
• and additionally optionally:
• b) an increased specific activity (Vmax [U/mg]) for DHCA in the enzymatic reduction of DHCA with NADPH as cofactor;
• c) a reduced substrate inhibition by DHCA;
• d) a modified cofactor specificity with respect to NADH and NADPH;
• and
• e) it being possible for these properties b), c) and d) to be present individually or in any combination.